Knockdown of dLKR/SDH potentiates dronc and ark transcription, and dLKR/SDH inhibits H3R17 methylation in vitro. (A) l(2)mbn cells were treated with dsRNA (RNAi) corresponding to mammalian Ndfip2 (control) or with dLKR/SDH and ethanol (0) or ecdysone (Ecd) for 6 or 24 h in triplicate. Levels of dronc and ark transcript were measured using real-time PCR and expressed relative to the internal housekeeping gene ribosomal protein rp49. Error bars represent SD. (B) 4 μg of calf thymus histones were incubated alone (−) or with 500 μg of recombinant GST-LKR (LKR) or GST-SDH (SDH) protein individually or together (LKR + SDH) for 4 h in the presence of NADH and α-ketoglutarate and were blotted with histone methylation–specific antibodies recognizing H3K4me1, H3K4me2, H3K4me3, H3K9me2, H3K9me3, and H3R17me2. H3R17me2 was assessed by methylating calf thymus histones first with CARMER before incubation with GST LKR/SDH. (C and D) 4 μg of calf thymus histones were incubated with increasing amounts of recombinant LKR (thrombin cleaved to remove GST) for 4 h. To the same reaction, 1 μg of either TRR or CARMER was added in methylation buffer (including ³H S-adenosyl methionine) for 90 min. Half of the total reaction was separated by SDS-PAGE and Coomassie stained, whereas the other half was electrophoresed, fixed, incubated in Amplify, dried, and exposed to hyperfilm. Methylation by TRR (H3K4me3) and CARMER (H3R17me2) are shown, as are total histones, dLKR/SDH, GST-TRR, and GST-CARMER. (E) Reactions performed as in C and D, including the addition of the SDH domain. Methylation by CARMER (H3R17me2) is shown, and Coomassie staining of histones in each sample is shown on the bottom panel.